ABSTRACT Sedentary behavior (SB) contributes to increased risk for obesity and metabolic disease, cognitive deficits, and affect disorders over the lifespan. These are critical outcomes because children with these risk factors are more likely to develop type 2 diabetes mellitus (T2DM). SB increases T2DM risk by promoting hyperglycemia and greater postprandial glycemic variability as well as via cognitive detriments and depressive symptoms that lead to poor energy balance behaviors, obesity, and worsening insulin resistance. Physical activity can reduce these risk factors, however less than half of US youth meet guideline recommendations, and physical activity continues to decline throughout adolescence. Thus, there is a critical need to test alternative intervention approaches to sustained bouts of exercise for the prevention of T2DM in children. We were the first to show that interrupting SB with short, 3-minute, bouts of moderate exercise improved glucose tolerance and negative mood in a single 3-hour session. However, it is unknown whether these short-term improvements translate to sustained multi-day benefits to metabolic, cognitive, and mood outcomes. Thus, the overall goal of this study is to test the efficacy of multi-day effects of interrupting SB as a T2DM prevention strategy in youth with overweight/obesity. We propose a Phase II RCT to compare the effects of SB interruptions vs. sustained bouts of exercise to prolonged sitting in 7-11-year-old children with overweight/obesity. This proposal will address the following aims: (1) determine the multi-day efficacy of interrupting sitting on glucose homeostasis measured by continuous glucose monitor and oral glucose tolerance tests; (2) determine the multi-day efficacy of interrupting sitting on cognitive function improvements; and (3) determine the multi-day efficacy of interrupting sitting on affect and anxiety improvements. This study is innovative because: (a) interrupting SB is a novel intervention strategy that has shown potential to acutely improve metabolic parameters, yet the longer-term effects are unknown and no prior studies have compared efficacy in reducing multiple T2DM risk factors using this approach vs. a single bout of exercise over multiple days in children; (b) the use of continuous glucose monitoring is a novel strategy to investigate multi- day glucose responses to SB interruptions and their association with cognitive and affective outcomes; and (c) investigating psychological responses to multiple days of interrupting SB vs. a single bout of exercise are novel outcomes that co-vary throughout the day, and are essential to elucidate if we are to develop novel intervention approaches that address factors associated withT2DM risk. Given the improvements in glucose homeostasis in our acute 3-hour trials, along with the dearth of pediatric studies investigating sustained interventions interrupting SB, this study is a significant and logical next step towards testing the efficacy of this approach for the reduction of multiple T2DM risk factors in children with overweight/obesity. Our approach is impactful because the rigorous, controlled lab setting will allow us to design stronger intervention strategies for children that can be translated to other settings, and age and weight groups, thereby contributing to efforts at reducing T2DM risk in U.S. youth.